LOCUS       sequence-472955-        5021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone to generate guideRNA shuttles, that also drives 
            expression of nuclear mKate in cardiomyocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5021)
  AUTHORS   Keeley S, Fernandez-Lajarin M, Bergemann D, John N, Parrott L, 
            Andrea BE, Gonzalez-Rosa JM
  TITLE     Rapid and robust generation of cardiomyocyte-specific crispants in 
            zebrafish using the cardiodeleter system.
  JOURNAL   Cell Rep Methods. 2025 Mar 24;5(3):101003. doi: 
            10.1016/j.crmeth.2025.101003.
  PUBMED    40132543
REFERENCE   2  (bases 1 to 5021)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2025-03-24"; volume: "5(3):101003. doi"; pages: " 
            10.1016/j.crmeth.2025.101003"
COMMENT     Sequence Label: pTol1-EcoRI-BmtI-BamHI-cmlc2-nmKate
FEATURES             Location/Qualifiers
     source          1..5021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    76..97
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(112..117,118..135,136..142)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    150..166
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     155..177
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     174..190
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     174..190
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1280..1300
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1301..1999
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHRV"
     regulatory      2001..2010
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2595..2612)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2595..2611)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2604..2626)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2820..2839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2939..2961
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2999..3017)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3085..3189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3190..3258,3259..4050)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3408..3427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4221..4809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4710..4729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4963..4980
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
       61 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      121 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      181 cagctatgac catgattacg ccaagcttgc atggtccctt tagccagtag cggttctagg
      241 cacgggccgt ccgggcggtg gcctggggcg gaaaactgaa ggggggcggc accggcggct
      301 cagcccttgt aatatattat atgcaccact attggtttac ttatgtcaca gtttgtaagt
      361 ttgtaacagc ctgaacctgg atcgaattca aagcccatcg tgctagcggt ccttagactg
      421 gatccctcca aatcagcaga cttaacattc tttaaaatga ttgattcaat agtgataaaa
      481 atcaggcata gccagttgta actttagata aattacagaa aatgtcaaat acagagaacc
      541 gattcttttt tatgatacat ccaagcacac atttaacaca atccaggcaa accccgaatt
      601 tcacagtcac aagcactgtt tgtacaagag ctttgcctaa ggacacacag tctctataag
      661 tccaggtcgt tggtttcact cttattttaa acatgtgaca tttttcctgc catcctgtct
      721 taggctgctg tttgcttcat tccatgtcac attaatttcc tcagtagcac cttttacaca
      781 cacagccaat cttttccaga aaattcaatt gctttgaaaa gataatgtgt gaacaaatcc
      841 atttagaaaa ggaaaattaa gaatttgtaa aatcatctgt aaattgttgg cattcttctg
      901 tatatgaaca tcacatcatt tacaggtaaa ggtctggtca ttaattatat gacaatttac
      961 tggtattatt ttgtgaaagg ggctattttc aatgcattca tccatccttt tcatccccca
     1021 aatctctcat tcatgtcccc ctccccatct gcacacttta tctcattttc caccctgctg
     1081 gaatctgagc acttgtgcag ttatcagggc tcctgtattt aggaggctct gggtgtccat
     1141 gtaggggacg aacagaaaca ctgcagacct ttatagaaga acaaatgata agagtcctca
     1201 tacataaaga ctccattaga aacgtcagtg acccaggagc ccagaccaac agcaaagcag
     1261 acagtgaacc gccaccatgc caaagaagaa gcgtaaggta gtgagcgagc tgattaagga
     1321 gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac
     1381 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga
     1441 gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa
     1501 aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct tccccgaggg
     1561 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     1621 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc
     1681 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagaccct
     1741 gtaccccgct gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
     1801 gggccacctg atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct
     1861 caagatgccc ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa
     1921 agagacctac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa
     1981 actggggcac agagtttaaa gccaccatgg agatctcgag actagtaatt aagtctcagc
     2041 caccgttaac tgaacatgtc aaaacctgtg gagactgttg agatttgatg ttctgaaaag
     2101 ataaagccta taaataaaat gttgcccaaa tttcctgcct gatgtttttc tttgtctttg
     2161 ctacatggct ttgctgctcg gatcggctca ctctgtgtat gccacgttca ctttgtactc
     2221 tccttctcac ggtaggttta ttatttttag atgtgcagtt agtttctgtg aaataacaca
     2281 ccacacactg atattgtctg tgcattgact tggtgagtgc acattgtttt tgatcttgac
     2341 atatttatat ttgattgatc aggtgaactg tgtgaatcta aagtgctcca tacagatgtt
     2401 ctgcattgaa aatattctca ttttattagt ggaagtgagt gtatgccagc ggccgcggta
     2461 ccctgcagtc tagacatttc agttgacgaa gacaaacaaa gttctgttgt gactatgggg
     2521 ggggggggcg cctggggatg gtctcgcccg gggagtaatt cagggtagaa ccgccactgc
     2581 ctttagcgac attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2641 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2701 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     2761 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     2821 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     2881 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2941 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     3001 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3061 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3121 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3181 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3241 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3301 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3361 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3421 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3481 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3541 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3601 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3661 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3721 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3781 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3841 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3901 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3961 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4021 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4081 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4141 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4981 gaagcggaag agcgcccaat acgcaaaccg cctctccccg c
//
